Evaluation of immunofluorescence techniques for detection of Cryptosporidium oocysts and Giardia cysts from environmental samples

Abstract

Cryptosporidium and Giardia species are enteric protozoa which cause waterborne disease. The detection of these organisms in water relies on the detection of the oocyst and cyst forms or stages. Monoclonal and polyclonal antibodies were compared for their abilities to react with Giardia cysts and Cryptosporidium oocysts after storage in water, 3.7% formaldehyde, and 2.5% potassium dichromate, upon exposure to bleach, and in environmental samples. Three monoclonal antibodies to Cryptosporidium parvum were evaluated. Each test resulted in an equivalent detection of the oocysts after storage, after exposure to bleach, and in environmental samples. Oocyst levels declined slightly after 20 to 22 weeks of storage in water, and oocyst fluorescence and morphology were dull and atypical. Oocyst counts decreased after exposure to 2,500 mg of sodium hypochlorite per liter, and fluorescence and phase-contrast counts were similar. Sediment due to algae and clays found in environmental samples interfered with the detection of oocysts on membrane filters. Two monoclonal antibodies and a polyclonal antibody directed against Giardia lamblia cysts were evaluated. From the same seeded preparations, significantly greater counts were obtained with the polyclonal antibody. Of the two monoclonal antibodies, one resulted in significantly lower cyst counts. In preliminary studies, the differences between antibodies were not apparent when used on the environmental wastewater samples. After 20 to 22 weeks in water, cyst levels declined significantly by 67%. Cysts were not detected with monoclonal antibodies after exposure to approximately 5,000 mg of sodium hypochlorite per liter. The enteric protozoa Giardia and Cryptosporidium species are well-documented causes of waterborne disease. From 1971 to 1985, Giardia species have been responsible for 92 outbreaks of disease associated with both animal and human fecal contamination of water supplies in the United States (2). Cryptosporidium species, newly recognized in 1980 as agents of diarrhea, have been documented in a number of outbreaks of waterborne disease in both the United States and the United Kingdom (4, 5, 8, 17). Zoonotic transmission has been demonstrated for Cryptosporidium parvum; therefore, many animals may also serve as sources of Cryptosporidium contamination of water supplies with the potential for human infection (3). Methods to study the occurrences of Cryptosporidium and Giardia species in water by detection of oocysts and cysts, respectively, have been developed. Large volumes of water are filtered, and the concentrate is examined by microscopic techniques. Developments in methods for Giardia and Cryptosporidium detection over the last few years have included the use of yam-wound filters for sample collection (9), various density gradients for sample clarification (15), and membrane filtration coupled with immunofluorescence techniques for cyst and oocyst detection and enumeration (10-16). Limitations of these methods include (i) poor recoveries influenced by water qualit...

The use of a ribosomal RNA targeted oligonucleotide probe for fluorescent labelling of viable Cryptosporidium parvum oocysts

“…The currently employed Cryptosporidium-specific mAbs recognize an antigen on the surface of oocysts that is present on both live and dead oocysts. Furthermore, the mAbs cross-react with Cryptosporidium species other than C. parvum (Rose et al 1989;Graczyk et al 1996) and bind to debris particles present in water samples (Vesey et al 1991;Campbell et al 1993) making detection and identification of oocysts difficult. In comparison, the Cry1 probe recognizes rRNA that is only present in viable oocysts, is potentially C. parvum-specific and does not bind to debris particles present in water samples.…”

“…Many of these outbreaks have been flow cytometry (Vesey et al 1993a(Vesey et al , 1994aHoffman et al 1997). However, the mAbs currently employed to detect C. parvum oocysts can react with both viable and non-viable oocysts and cross-react with Cryptosporidium species that are not infectious to humans (Rose et al 1989;Graczyk et al 1996). Therefore, immunofluorescence alone does not discriminate C. parvum from other Cryptosporidium species, nor is the viability of oocysts determined.…”

Pathogens, faecal indicators and human-specific microbial source-tracking markers in sewage

“…Regular microscopy is the preferred method for detecting multicellular parasites for which a digital imaging system to identify and quantify several species of helminth eggs has recently been developed (Maya et al 2006;Jim enez et al 2016). Advanced methods such as immunofluorescence microscopy (Rose et al 1989), flow cytometry (Vesey et al 1994) or laser scanning cytometry (Montemayor et al 2007) are preferred for protozoa.…”

An immunomagnetic separation polymerase chain reaction assay for rapid and ultra-sensitive detection of Cryptosporidium parvum in drinking water

“…In addition to its high sensitivity, this IMS-PCR assay is rapid, providing results in less than 24 h. By comparison, the recommended methods for Cryptosporidium analysis [4] include concentration of large volumes of water (100^500 l) using cartridge ¢lters, separation from background debris by £otation on a Percoll-sucrose gradient and then analyzing a proportion of the ¢nal concentrate by immuno£uorescence detection [12]. This is tedious, time-consuming (at least 48 h) and shows poor recovery (30^40%) [13].…”